Long term culture of mouse hybridoma HB8852 cells in a protein free medium

Summary We developed a protein free medium based on ERDF medium containing 80 M FeSO₄, ethanolamine and selenite. Although this medium contained neither transferrin nor insulin, the medium could support long term culture of mouse hybridoma HB8852 without any significant changes in antibody production.     

Purification and characterization of aβ-galactosidase fromTreponema phagedenis (Reiter strain)

A β-galactosidase was highly purified from a cellular extract ofTreponema phagedenis (Reiter strain) by ammonium sulfate precipitation and successive chromatography on Sepharose 6B and DEAE-Sephadex. The purified enzyme was homogeneous in SDS-polyacrylamide gel electrophoresis. The molecular weight estimated was 580,000. The optimal pH, ionic strength, and temperature were 6.5, 0.1, and 50°C, respecitvely. The enzyme was stable only at around pH 6.5 and at temperatures lower than 35°C. The enzyme was irreversibly inhibited byp-chloromercuribenzoate and divalent cations such as Hg²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺. The Km values forp-nitrophenyl-β-D-galactopyranoside,o-nitrophenyl-β-D-galactopyranoside, and lactose were 0.29, 0.36, and 5.4 mM, respectively.     

A method of statistical neurodynamics

A method of statistical neurodynamics is presented for treating ensembles of nets of randomly connected neuron-like elements. The concept of a macrostate plays a fundamental role in statistical neurodynamics and a criterion is given for ascertaining that given macroscopic quantities together constitute a macrostate. The activity of a nerve net is shown to be a macrostate and the equation of the dynamics of the activity is elucidated for various ensembles of random nerve nets. It is shown that the distance between two microstates can also be treated as a macrostate in a generalized sense. The equation of its dynamics represents how the distance between two states changes in the course of state transitions. The dynamics of distance reveals interesting microscopic properties of random nerve nets, such as the stability of state-transition, the transient lengths, etc.     

Dynamics of pattern formation in lateral-inhibition type neural fields

The dynamics of pattern formation is studied for lateral-inhibition type homogeneous neural fields with general connections. Neural fields consisting of single layer are first treated, and it is proved that there are five types of pattern dynamics. The type of the dynamics of a field depends not only on the mutual connections within the field but on the level of homogeneous stimulus given to the field. An example of the dynamics is as follows: A fixed size of localized excitation, once evoked by stimulation, can be retained in the field persistently even after the stimulation vanishes. It moves until it finds the position of the maximum of the input stimulus. Fields consisting of an excitatory and an inhibitory layer are next analyzed. In addition to stationary localized excitation, fields have such pattern dynamics as production of oscillatory waves, travelling waves, active and dual active transients, etc.This research was supported in part by a Sloan Foundation grant to the Center for Systems Neuroscience, University of Massachusetts at AmherstOn sabbatical leave from the University of Tokyo     

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named “Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides” were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability.     

Continuous Presence of Noroviruses and Sapoviruses in Raw Sewage Reflects Infections among Inhabitants of Toyama,Japan (2006 to 2008)

Various genotypes of norovirus (NoV) (genogroup I genotype 1 [GI.1], -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15), and sapovirus (SaV) (GI.1 and GI.2, GII.1, and GIV.1) were detected from raw sewage from April 2006 to March 2008, while limited numbers of genotypes of NoV (GI.8, GII.4, GII.6, and GII.13) and SaV (GII.3 and GIV.1) and of NoV (GII.4, GII.7, and GII.13) were detected from clinical cases and healthy children, respectively. During the winter 2006 to 2008, a large number of sporadic gastroenteritis outbreaks and many outbreaks caused by NoV GII.4 occurred among inhabitants in Toyama, Japan. The copy number of genomes of NoV GII detected from raw sewage changed in relation to the number of outbreaks. NoV strains of the same genotypes observed in both raw sewage and human specimens belonged to the same cluster by phylogenetic analysis and had almost identical nucleotide sequences among each genotype. These data suggest that NoVs and SaVs detected from raw sewage reflect the viruses circulating in the community, irrespective of symptoms, and that subclinical infections of NoV are common in Japan. Combined surveys of raw sewage with those of clinical cases help us to understand the relationship between infection of these viruses and gastroenteritis.Norovirus (NoV) and sapovirus (SaV), members of the Caliciviridae family, are considered to be a major cause of acute gastroenteritis in humans. Both NoV and SaV infect humans via the fecal-oral route and cause family or community-wide outbreaks, mainly in the winter season. NoVs are shed in feces at a level of 10⁵ to 10⁹ virus particles per gram during the symptomatic phase (32, 37), and viruses are continuously shed from patients after cessation of the symptoms (28, 37, 40). In addition, recent reports showed relatively high levels of shedding of the viruses from asymptomatic individuals (7, 8, 32, 37).NoVs and SaVs show high diversity in their genomes (5, 9). According to such a genetic diversity, they are classified into several genogroups (genogroup I [GI], GII, and GIV for human NoV and GI, GII, GIV, GV for human SaV) and further divided into many genotypes (NoV GI genotypes 1 to 14 [GI.1-14] and GII.1-17 and SaV GI.1-5, GII.1-6, GIV.1, and GV.1) (10, 17, 18). In 2006 to 2007, NoV GII.4 caused a large number of outbreaks of acute gastroenteritis worldwide (1, 11, 35, 43, 45). However, the other genotypes of NoV and SaV may infect humans asymptomatically and persist in the environment.Raw sewage could contain enteric viruses shed from affected people, and therefore, detectable viruses in raw sewage would reflect the actual state of the circulating viruses in the area. We previously reported that polioviruses in raw sewage and river water were isolated at the same time as oral vaccination in babies, and these isolates were derived from vaccine strains (13, 30). We also showed that the nucleotide sequences of echovirus type 13 isolated from river water were closely related to those from patients with aseptic meningitis during the outbreak in 2002 (14). For NoVs and SaVs, many epidemiological surveys have been conducted to determine the prevalence and virological properties of these viruses (42). Previous reports have shown that the nucleotide sequences of NoV strains from stools of outbreaks in nursing homes and from sewage were identical for an individual outbreak (26), and NoVs detected from gastroenteritis patients, domestic sewage, river water, and cultivated oysters in the area were related to each other (44). However, less is known about infection of the viruses with minor genotypes that are silently circulating in the population.In this study, we investigated NoVs and SaVs in raw sewage from 2006 to 2008 in Japan and compared the results with the viruses detected from clinical cases as well as healthy individuals to show the comprehensive prevalence of these viruses in the community.     

A circa-decadal change in the gastropod fauna on a tidal flat in an island mangrove estuary

An investigation on gastropod fauna was carried out on a tidal flat in the Nagura Estuary on Ishigaki Island, the Ryukyu Islands in 1989 and 1998 using similar methods. 470-480 quadrats covering ca. 1900 m2 were surveyed during low tides from February to April in each year. Of the total 19 species recorded, the ranges of eight species had varied significantly between the two surveys, with six species expanding their range and two species contracting their range. Percentage in abundance of muddy-bottom species and tropical (<29 degrees N) species increased significantly between the two years. Topography of the flat also changed: the mouth of the river was narrowed and the elevated sections of the tidal flat expanded. During the period from 1984 to 1998, the farmland development around the study site caused influxes of soil into the estuary and the sea-water temperature was rising. These results suggest that the topographical changes due to soil influx and the rising temperature affected the gastropod assemblage at the study site, by increasing the abundance of muddy-bottom species and tropical species. The methodology used in this study, i.e. surface observation at low tides, includes more than 95% of the gastropod fauna, demonstrating the usefulness of surface counts for the study of soft-bottom fauna.     

Isothermal acid-titration calorimetry for evaluating the pH dependence of protein stability

A new method, which can be called as isothermal acid-titration calorimetry (IATC), was proposed for evaluating the enthalpy of protein molecules as a function of pH using isothermal titration calorimetry (ITC). This measurement was used to analyze the acid-denaturation of bovine ribonuclease A. The enthalpy change by acid-denaturation of this protein was estimated as 310 kJ/mol at pH 2.8 and 40 degrees C. This value agreed well with the enthalpy change obtained by differential scanning calorimetry. The midpoint pH and proton binding-number difference observed by IATC agreed well with those of the acid transition of the three-dimensional structure monitored by circular dichroism spectrometry. The van't Hoff enthalpy of the transition was derived from the temperature dependence of the midpoint pH and the proton binding-number difference. It agreed well with the calorimetric enthalpy change directly observed by IATC, strongly indicating that there was no stable intermediate state during the acid transition of this protein.